Urolithin A improves motility, antioxidant defense, and mitochondrial function of chilled canine spermatozoa during 72-h liquid storage

Dog semen conservation at cold temperatures (5 °C) is a valuable reproductive management tool, but it is bedeviled by issues of oxidative stress and mitochondrial damage that reduce sperm quality and viability. Urolithin A (UA), a natural metabolite derived from gut microbiota, has been found to be a molecule possessing strong antioxidant and mitochondrial protective activities. The objective of the study was to evaluate the protective effect of UA on the quality of dog sperm preserved in liquid form at 5 °C for 72 h. Semen samples were extended using a Tris-egg yolk extender supplemented with different concentrations of UA (0, 1, 5, and 10 µM). Evaluations were performed at 0, 24, 48, and 72 h of storage. The results showed that supplementation with 1 and 5 µM UA significantly preserved the total motility, progressive motility, and kinematic parameters (VCL, VSL, VAP). Furthermore, these doses significantly preserved sperm viability, plasma membrane integrity, and DNA integrity compared to the control group. Biochemically, UA enhanced the total antioxidant capacity (TAC), elevated the activity of key antioxidant enzymes (SOD, CAT, GPx), and reduced the markers of oxidative stress (MDA and H₂O₂). Concurrently, UA treatment preserved mitochondrial membrane potential (MMP), oxygen consumption rate (OCR), and intracellular ATP content. The 10 µM concentration was not effective in providing protective effects and acted similarly to the control. In conclusion, Urolithin A, particularly at an optimal concentration of 1 µM, may be capable of alleviating oxidative damage and preserving mitochondrial function, thereby being a potent supplement for canine semen extenders to enhance sperm quality and viability during refrigerated storage.